NodS

NodS is a bacterial methyltransferase involved in the pathway leading to the biosynthesis of Nod factor, an important signaling molecule released by rhizobial bacteria in the course of endosymbiotic interaction with legumes. NodS from Bradyrhizobium japonicum WM9 is the first S-adenosyl-L-methionine-dependent methyltransferase specific for chitooligosaccharide substrates, the structure of which has been solved. The structures with (3ofk) and without (3ofj) S-adenosyl-L-homocysteine ligand, which is chemically very similar to the methyl donor used by NodS, S-adenosyl-L-methionine, are available.

Rhizobia-legumes symbiosis
The endosymbiotic relationship between nitrogen-fixing rhizobial bacteria and certain plants (legumes) is of the utmost importance for the nitrogen cycle in the biosphere and hence an object of intense study by specialists from various areas of biology. What is more, although the use of artificial nitrogen-containing fertilizers made it possible to bypass the strict dependence on this process, its understanding is still crucial for the efficient agriculture. Biological nitrogen fixation requires nitrogenase, an enzyme present in, among others, rhizobial bacteria but absent in plants. The rhizobia are a diverse range of soil bacteria. It is thought that after the symbiotic capabilities were first acquired by some of them, they spread by means of horizontal transfer of a plasmid or genomic island containing genes important for the process. The symbiotic relationship between the two symbionts depends on the formation of invasion structures (nodules) by the host, allowing bacteria to enter the plant root, colonize a specific type of cells and be transformed into bacteroids which perform nitrogen fixation. As complementarity between various signals and their cognate receptors is required for the efficient symbiosis, its emergence most likely required coevolution of plants and bacteria. One of the bacterial nodulating symbionts is Bradyrhizobium japonicum WM9, which is involved in a partnership with lupine and serradella legumes.

Chemical signals
The very first stage of this process is an exchange of chemical signals between the symbionts. On sensing a flavonoid compound released by the plant, the bacteria produce a signalling molecule known as Nod factor, which is a lipochitooligosaccharide derivative. This molecule in turn induces nodule formation by the plant. Many types of flavonoids and Nod factors are known and which ones are present influences the specificity of the interaction. It must be mentioned that while these two molecules are important signals and specificity determinants, there are numerous others.

Nod factor synthesis
Nod factor synthesis involves the coordinated action of several rhizobial enzymes encoded by the nodulation-specific genes. NodC, NodB and NodA, found in nearly all rhizobial strains, synthesize the main part of Nod factor's structure, while several others, including NodS, NodU, NolO, NodL, and NolL, introduce species- and strain-specific modifications, such as N-methylation, carbamoylation, acetylation, fucosylation etc.

Structure and function of NodS


Fold
NodS is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates the deacetylated nitrogen atom at the nonreducing end of the chitooligosaccharide substrate, converting at the same time the S-adenosyl-L-methionine methyl donor into S-adenosyl-L-homocysteine, which is then released as a by-product (see figure above). S-adenosyl-L-methionine-dependent methyltransferases, many of which have been reported to date, methylate a wide variety of substrates and are involved in diverse processes ranging from biosynthetic pathways to gene silencing. Those of them which have been structurally analyzed have been divided into five structural families known as classes I–V, class I, to which NodS belongs, being the most numerous. Its typical fold consists of a seven-stranded β-sheet (the strands are named 1-7 beginning from the N-terminus) with a reversed β-hairpin (residues 182-189) at the C-terminal end of the sheet, between strands β6 and β7. The sheet, which is characterized by a curvature due to the presence of three β-bulges, is surrounded on both sides by α-helices (named A-G beginning from the N-terminus), forming an α/β/α folding pattern. In the apo form shown here, only 6 out of 7 helices are visible. In addition to conventional helices, there are also three short, 310 helices (not represented as helices), each one-turn-long (residues 19-21, 63-65, 117-119), two of which are visible in the apo structure. The fact that strand β7 is inserted in an anti-parallel orientation results in a divergence from a classic Rossmann fold, to which the structure is otherwise classifiable.

Ligand binding
During the NodS-catalyzed reaction, the chitooligosaccharide substrate is methylated while the methyl donor, S-adenosyl-L-methionine, is transformed into S-adenosyl-L-homocysteine. The latter product is, however, chemically still highly similar to S-adenosyl-L-methionine and hence the structure of NodS bound to S-adenosyl-L-homocysteine gives reliable representation of the binding mode of both S-adenosyl-L-homocysteine and, by inference, S-adenosyl-L-methionine to the enzyme. Notice the position of the helix A (the most N-terminal), which was not visible in the apo form, but got structured upon ligand binding; this helix, unlike others, is positioned on top of the β-sheet structure, not on its side, and is in close proximity to the ligand. There are many other small changes in the structure of ligand-bound NodS when compared to the apo form, mainly around the binding cavity, but the deviations do not exceed 0.9 Å. The S-adenosyl-L-homocysteine -binding cavity is predominately hydrophobic, but numerous polar interactions are likely to play a role in positioning the ligand in its specific orientation. There are 11 polar interactions between NodS residues and S-adenosyl-L-methionine ligand and around 5-7 additional ones between the water molecules present in the binding pocket and the ligand. The amino group of the homocysteine amino acid moiety is anchored by the main-chain carbonyl groups of Gly 52 and Ala 114 from loop β1–αC and strand β4, respectively. The carboxy group of the same moiety interacts with the side chains of the basic amino acid residues Arg 31 and His 32 in helix αB. The hydroxyl groups of the ribose fragment of S-adenosyl-L-homocysteine form a pair of forked hydrogen bonds with both oxygen atoms of the carboxylic group of Asp 73 and a single hydrogen bond with main-chain nitrogen of Ala 54, Asp 73 being located at the tip of the β2 strand and Ala 54 in loop β1–αC. Finally, <scene name='NodS/Domek/8'>the adenine ring of the ligand is interacting with the main-chain nitrogen of Ile 99 and by the side chain of Asp 98, both from the β3–β4 loop. This adenine ring is also capable of making CH-π interactions with aliphatic side chains of valines 74 and 116. <scene name='NodS/Domek/9'>An additional interaction can potentially be formed between the sulfonium group of the ligand and the π system of Trp 20 when the methyl donor, S-adenosyl-L-methionine is bound instead of the homocysteine derivative. This interaction is likely to contribute to the larger affinity of NodS towards the methyl donor (a substrate) comparing to the homocysteine derivative (a products). The overall positioning of the methyl donor inside the protein is such that it is <scene name='User:Marcin_Jozef_Suskiewicz/Sandbox_MarcinSuskiewicz/4/1'>guarded from the environment, but the fragment of it where the methyl group of S-adenosyl-L-methionine is located <scene name='User:Marcin_Jozef_Suskiewicz/Sandbox_MarcinSuskiewicz/4/2'>is exposed to the putative substrate-binding canyon. The canyon is roughly 22 Å long, 10.5 Å wide and 10.5 Å deep. It is located near the N-terminal end of helix αB and the C-terminal end of strand β4. No structure of NodS with the substrate bound is available, but computational docking suggests that this canyon is indeed an energetically favourable binding site, mainly due to a number of possible polar interactions.

</StructureSection>